Mutation of amphioxus Pdx and Cdx demonstrates conserved roles for ParaHox genes in gut, anus and tail patterning.

BACKGROUND: The homeobox genes Pdx and Cdx are widespread across the animal kingdom and part of the small ParaHox gene cluster. Gene expression patterns suggest ancient roles for Pdx and Cdx in patterning the through-gut of bilaterian animals although functional data are available for few lineages. To examine evolutionary conservation of Pdx and Cdx gene functions, we focus on amphioxus, small marine animals that occupy a pivotal position in chordate evolution and in which ParaHox gene clustering was first reported. RESULTS: Using transcription activator-like effector nucleases (TALENs), we engineer frameshift mutations in the Pdx and Cdx genes of the amphioxus Branchiostoma floridae and establish mutant lines. Homozygous Pdx mutants have a defect in amphioxus endoderm, manifest as loss of a midgut region expressing endogenous GFP. The anus fails to open in homozygous Cdx mutants, which also have defects in posterior body extension and epidermal tail fin development. Treatment with an inverse agonist of retinoic acid (RA) signalling partially rescues the axial and tail fin phenotypes indicating they are caused by increased RA signalling. Gene expression analyses and luciferase assays suggest that posterior RA levels are kept low in wild type animals by a likely direct transcriptional regulation of a Cyp26 gene by Cdx. Transcriptome analysis reveals extensive gene expression changes in mutants, with a disproportionate effect of Pdx and Cdx on gut-enriched genes and a colinear-like effect of Cdx on Hox genes. CONCLUSIONS: These data reveal that amphioxus Pdx and Cdx have roles in specifying middle and posterior cell fates in the endoderm of the gut, roles that likely date to the origin of Bilateria. This conclusion is consistent with these two ParaHox genes playing a role in the origin of the bilaterian through-gut with a distinct anus, morphological innovations that contributed to ecological change in the Cambrian. In addition, we find that amphioxus Cdx promotes body axis extension through a molecular mechanism conserved with vertebrates. The axial extension role for Cdx dates back at least to the origin of Chordata and may have facilitated the evolution of the post-anal tail and active locomotion in chordates.

Developmental biology meets toxicology: contributing reproductive mechanisms to build adverse outcome pathways.

An adverse outcome pathway (AOP) is a simplified description of the sequence of mechanistic events that lead to a particular toxicological effect, from initial trigger to adverse outcome. Although designed to inform regulatory risk assessors, the AOP framework also provides a platform for innovative collaborations between experts from relevant research fields and the regulatory community. The underpinning for any AOP is basic knowledge about molecular and developmental processes; such knowledge can only be attained by solid bioscientific research. Starting with this fundamental knowledge, the objective is to devise novel testing strategies that focus on key events in a causative pathway. It is anticipated that such a knowledge-based approach will ultimately alleviate many of the burdens associated with classical chemical testing strategies that typically involve large-scale animal toxicity regimens. This hails from the notion that a solid understanding of the underlying mechanisms will allow the development and use of alternative test methods, including both in vitro and in silico approaches. This review is specifically targeted at professionals working in bioscientific fields, such as developmental and reproductive biology, and aims to (i) inform on the existence of the AOP framework and (ii) encourage new cross-disciplinary collaborations. It is hoped that fundamental biological knowledge can thus be better exploited for applied purposes: firstly, an improved understanding of how our perpetual exposure to environmental chemicals is causing human reproductive disease and, secondly, new approaches to screen for harmful chemicals more efficiently. This is not an instructional manual on how to create AOPs; rather, we discuss how to harness fundamental knowledge from the biosciences to assist regulatory toxicologists in their efforts to protect humans against chemicals that harm human reproductive development and function.

Fetal anogenital distance using ultrasound.

OBJECTIVE: This study measured anogenital distance (AGD) during late second/early third trimester of pregnancy to confirm previous findings that AGD can be measured noninvasively in the fetus using ultrasound and further showed differences in reference ranges between populations. METHOD: Two hundred ten singleton pregnancies were recruited at the Rosie Hospital, Cambridge, UK. A 2D ultrasound was performed between 26 and 30 weeks of pregnancy. AGD was measured from the centre of the anus to the base of the scrotum in males and to the posterior convergence of the fourchette in females. RESULTS: A significant difference in AGD between males and females (P < .0001) was found, replicating previous results with a significant correlation between estimated fetal weight (EFW) and AGD in males only (P = .006). A comparison of AGD using reference data from an Israeli sample (n = 118) and our UK sample (n = 208) showed a significant difference (P < .0001) in both males and females, after controlling for gestational age (GA). CONCLUSION: Our results confirm that AGD measurement in utero using ultrasound is feasible. In addition, there are strong sex differences, consistent with previous suggestions that AGD is influenced by prenatal androgen exposure. AGD lengths differ between the UK and Israel; therefore, population-specific normative values may be required for accurate clinical assessments.

Prenatal Evaluation for Detection of Anorectal Atresia: Value of Ultrasound.

OBJECTIVES: To investigate the applicability and value of ultrasound (US) in the diagnosis of anorectal atresia. METHODS: Between January 2008 and January 2016, we prospectively evaluated 63,101 fetuses (gestational age, 20-38 weeks), including low- and high-risk populations using 2-dimensional US scans. An abnormal imaging finding was defined as an anal canal diameter of less than the 95% confidence interval (small anal canal) of the normal range or the absence of an anal canal and rectum. Imaging findings were considered normal on detection of an anal canal with a normal width and the absence of abnormalities. Prenatal imaging findings were confirmed by a postnatal or postmortem examination. RESULTS: Among the investigated fetuses, 28 showed evidence of anorectal atresia on US scans, and 22 of those with anorectal atresia had additional anomalies. Six cases of isolated anorectal atresia were successfully detected during the preclusive prenatal US scans. Four cases of a low imperforate anus (including 2 covered anuses) yielded false-negative results, indicating a diagnostic rate of 87.5% (28 of 32). The normal appearance of the fetal rectum and anal canal ruled out anorectal atresia in 30 fetuses with a dilated colon. Additionally, there were 3 false-positive cases, in which a narrow anal canal was observed. CONCLUSIONS: Identifying the abnormal appearance or absence of the fetal anal canal and rectum on preclusive US anomaly scans is useful for prenatal diagnosis or exclusion of anorectal atresia, which may help improve the detection of isolated anorectal atresia. Furthermore, a combined evaluation of the longitudinal and axial appearances of the fetal anal canal and rectum can improve diagnostic accuracy.

Prenatal exposure to persistent organic compounds and their association with anogenital distance in infants.

RESEARCH QUESTION: What is the association between prenatal exposure to persistent organic pollutants, separately and combined, and anogenital distance (in-utero endocrine disruption marker). DESIGN: A cohort study conducted in Sonora, Mexico. Blood concentrations of polychlorobiphenyls (PCB) 28, 74, 118, 138/158, 153, 170, 180 and the isomers of dichlorodiphenyltrichloroethane (DDT) and its metabolites were determined in women in the third trimester of pregnancy; three variants of anogenital distance were measured on five occasions during the first year of life of their infants: 82 girls (402 observations) and 74 boys (356 observations). RESULTS: Boys had negative and significant associations between anogenital distance/height and the concentrations of PCB 28 (beta = - 0.005;P = 0.006), PCB 74 (beta = - 0.003;P = 0.013), and PCB 170 (beta = - 0.005;P = 0.001) when analysed individually. Negative and significant associations were also found using statistical models applied to mixtures of compounds. The latter associations were sometimes larger in magnitude and significance, suggesting a possible potentiation of the compounds. No associations were observed between anogenital distance and DDT in either sex or with PCB in girls. CONCLUSIONS: The decreased anogenital distance associated with prenatal exposure to the persistent organic pollutants, observed consistently in different analyses, suggests an under-masculinizing effect of these environmental pollutants in boys.

First trimester expression of anorectal malformation: Case report and review of the literature.

A 26-years-old woman, underwent an ultrasound examination at 13.4 weeks. A cystic structure was identified in the right lower abdomen. Gradually, the cystic mass was replaced by echogenic content and eventually attained the appearance of hyperechoic bowel. At 21.2 weeks, the anal sphincter could not be demonstrated which was consistent with the diagnosis of isolated anal agenesis. Amniocentesis revealed 46XY karyotype with normal comparative genomic hybridization. After termination of pregnancy at 23 weeks, an autopsy revealed an isolated high type anorectal malformation (ARM) without fistula. We reviewed all 14 cases reported in the literature of first trimester sonographic expression of ARM.

Clarification of mammalian cloacal morphogenesis using high-resolution episcopic microscopy.

The developmental process through which the cloaca transforms from one hollow structure to two separated urinary and digestive outlets remains controversial and speculative. Here, we use high-resolution episcopic microscopy to examine a comprehensive series of normal and mutant mouse cloaca in which the detailed 3-dimensional (3-D) morphological features are illuminated throughout the development. We provide evidence that the dorsal peri-cloacal mesenchyme (dPCM) remains stationary while other surrounding tissues grow towards it. This causes dramatic changes of spatial relationship among caudal structures and morphological transformation of the cloaca. The 3-D characterizations of Dkk1 mutants reveal a hyperplastic defect of dPCM, which leads to a significant anterior shift of the caudal boundary of the cloaca, premature occlusion of the cloaca and, imperforate anus phenotype. Conversely, Shh knockout causes a severe hypoplastic defect of cloaca mesenchyme including dPCM and persistent cloaca. Collectively, these findings suggest that formation of the dPCM is critical for cloacal morphogenesis and furthermore, growth and movement of the mesenchymal tissues towards the dPCM lead to the cloaca occlusion and separation of the urinary and digestive outlets.

Transient Distention of Right Posterior Located Sigma, a New Sonographic Sign for the Prenatal Diagnosis of Anal Atresia.

We describe a new sonographic sign for the detection of anal atresia in the early midtrimester on transvaginal sonography. In six cases of fetal anal atresia, the finding of a transient, distended, and right-sided sigmoid colon was observed at 13-16 weeks' gestation. Three cases have undergone pregnancy termination due to multiple anomalies. In the other three, the colonic distension resolved by 19 weeks' gestation. In two of these, the finding was isolated, and no other anomalies were detected. In all six cases, anal atresia or cloaca was confirmed on postabortal autopsy or after delivery. © 2016 Wiley Periodicals, Inc. J Clin Ultrasound 45:160-162, 2017.

Developmental alterations of the C. elegans male anal depressor morphology and function require sex-specific cell autonomous and cell non-autonomous interactions.

We studied the Caenorhabditis elegans anal depressor development in larval males and hermaphrodites to address how a differentiated cell sex-specifically changes its morphology prior to adulthood. In both sexes, the larval anal depressor muscle is used for defecation behavior. However in the adult males, the muscle's sarcomere is reorganized to facilitate copulation. To address when the changes occur in the anal depressor, we used YFP:actin to monitor, and mutant analysis, laser-ablation and transgenic feminization to perturb the cell's morphological dynamics. In L1 and L2 stage larva, the muscle of both sexes has similar sarcomere morphology, but the hermaphrodite sex-determination system promotes more growth. The male anal depressor begins to change in the L3 stage, first by retracting its muscle arm from the neurons of the defecation circuit. Then the muscle's ventral region develops a slit that demarcates an anterior and posterior domain. This demarcation is not dependent on the anal depressor's intrinsic genetic sex, but is influenced by extrinsic interactions with the developing male sex muscles. However, subsequent changes are dependent on the cell's sex. In the L4 stage, the anterior domain first disassembles the dorsal-ventral sarcomere region and develops filopodia that elongates anteriorly towards the spicule muscles. Later, the posterior domain dissembles the remnants of its sarcomere, but still retains a vestigial attachment to the ventral body wall. Finally, the anterior domain attaches to the sex muscles, and then reassembles an anterior-posteriorly oriented sarcomere. Our work identifies key steps in the dimorphic re-sculpting of the anal depressor that are regulated by genetic sex and by cell-cell signaling.

Fetal growth of the anal sinus and sphincters, especially in relation to anal anomalies.

PURPOSE AND METHODS: The anal sinuses, small furrows above the pectinate line, sometimes form perianal abscesses in adults. We examined the pattern of fetal growth of the anal sinus and sphincters using 22 mid-term (8-18 weeks) and 6 late-stage (30-38 weeks) fetuses. RESULTS: In mid-term fetuses, the external and internal sphincters gradually increased in thickness, depending on specimen size (from 0.2 to 1.5 mm), whereas the anteroposterior diameter of the anal canal at the epithelial junction was relatively stable (0.5-1.0 mm) irrespective of specimen size. Anal canal diameter increased less than twofold between mid-term and late-stage fetuses, from 0.5-1.0 to almost 2 mm, whereas sphincter thickness increased over tenfold, from 0.2-1.5 to almost 3.5 mm. The anal sinus often showed balloon-like enlargement when the sphincter muscle bundles were tightly packed in mid-term, but not in late-stage fetuses. CONCLUSIONS: Large concentric mechanical stress from the sphincters in late-stage fetuses apparently prevented the anal sinus from expanding in a balloon-like manner. Conversely, to avoid anal stenosis, the growing sinuses maintained a luminal space of the anal canal in response to stress from rapidly growing sphincters. The inferiorly extending sinus usually provided temporal double canals separated by a thick column. In the presence of double lumens, anal canal duplication is likely to develop without any abnormalities of the anal epithelium and sphincters.

Anatomical basis of the coordination between smooth and striated urethral and anal sphincters: loops of regulation between inferior hypogastric plexus and pudendal nerve. Immuno-histological study with 3D reconstruction.

PURPOSE: (1) Describe both nervous pathways to the sphincters, and highlight the anatomical support of their coordination. (2) Obtain a 3D representation of this complex innervation system. METHODS: A computer-assisted anatomical dissection technique was used. Serial histological sections were cut in the pelvis of four female human foetuses (aged 19-32 weeks of gestation). The sections were treated with conventional staining, and with seven different immunostainings. The sections were digitalized and, finally, a 3D representation was built from the corresponding images. RESULTS: Myelinated and sensory fibres were detected at the inferior hypogastric plexus (IHP) level. Our analysis showed that most of the afferent sensory fibres come from the urinary and anal sphincters through the anterior and posterior branches of the IHP respectively. A highly positive nitrergic (anti-NOS1) and sensitive (anti-CGRP) labelling was found in the external layer of the urethral sphincter. The 3D representation allowed describing the two components of the innervation system. A sensory-motor regulation loop was found for both sphincters. CONCLUSION: A 3D description of the components of both nervous pathways to the sphincters has been established. Our findings on the innervation of the sphincters tend to question the classical infra/supra levatorian muscle description. The coordinated work of the internal and external layers of the anal and urethral sphincter is probably mediated by multiple roles regulation.

Dkk1 in the peri-cloaca mesenchyme regulates formation of anorectal and genitourinary tracts.

Anorectal malformation (ARM) is a common birth defect but the developmental history and the underlying molecular mechanism are poorly understood. Using murine genetic models, we report here that a signaling molecule Dickkopf-1 (Dkk1) is a critical regulator. The anorectal and genitourinary tracts are major derivatives of caudal hindgut, or the cloaca.Dkk1 is highly expressed in the dorsal peri-cloacal mesenchymal (dPCM) progenitors. We show that the deletion of Dkk1 causes the imperforate anus with rectourinary fistula. Mutant genital tubercles exhibit a preputial hypospadias phenotype and premature urethral canalization.Dkk1 mutants have an ectopic expansion of the dPCM tissue, which correlates with an aberrant increase of cell proliferation and survival. This ectopic tissue is detectable before the earliest sign of the anus formation, suggesting that it is most likely the primary or early cause of the defect. Deletion of Dkk1 results in an elevation of the Wnt/ß-catenin activity. Signaling molecules Shh, Fgf8 and Bmp4 are also upregulated. Furthermore, genetic hyperactivation of Wnt/ß-catenin signal pathway in the cloacal mesenchyme partially recapitulates Dkk1 mutant phenotypes. Together, these findings underscore the importance ofDKK1 in regulating behavior of dPCM progenitors, and suggest that formation of anus and urethral depends on Dkk1-mediated dynamic inhibition of the canonical Wnt/ß-catenin signal pathway.

Temporal and spatial expression of caudal-type homeobox gene-2 during hindgut development in rat embryos with ethylenethiourea-induced anorectal malformations.

The main aim of this study was to determine Cdx2 expression patterns during anorectal development in normal and anorectal malformation (ARM) embryos with a view to establishing the possible role of Cdx2 in ARM pathogenesis. ARM was induced with ethylenethiourea on the 10th gestational day (GD10) in rat embryos, and Cesarean deliveries were performed to harvest the embryos. The temporal and spatial expression of Cdx2 was evaluated in normal rat embryos (n = 303) and ARM embryos (n = 321) from GD13 to GD16. Immunohistochemical staining revealed that, in normal embryos, Cdx2 was mainly expressed on the epithelium of the urorectal septum (URS) and the hindgut on GD13. On GD14, Cdx2-immunopositive cells were extensively detected on the URS, hindgut, and cloacal membrane. On GD15, increased immunopositive tissue staining on the anal membrane was evident. In ARM embryos, the epithelium of the cloaca, URS, and anorectum were negative or faintly immunostaining for Cdx2. Analyses by Western blot and real-time reverse transcription plus the polymerase chain reaction revealed that, in the normal group, Cdx2 protein and mRNA expression showed time-dependent changes in the developing hindgut from GD13 to GD16. Upon the URS division of the cloaca into the primitive rectum and urogenital sinus (UGS) on GD15, Cdx2 expression began to decrease. Moreover, the Cdx2 expression level in the ARM group from GD13 to GD14 was significantly lower than that in the normal group (P < 0.05). In ARM embryos, an imbalance in the spatiotemporal expression of Cdx2 was noted during anorectal morphogenesis from GD13 to GD16. Downregulation of Cdx2 at the time of cloacal separation into the primitive rectum and UGS might thus be related to the development of ARM.

Acoel development indicates the independent evolution of the bilaterian mouth and anus.

Most bilaterian animals possess a through gut with a separate mouth and anus. It is commonly believed that during the transition from radial to bilateral symmetry, both openings evolved simultaneously by the lateral closure of a slit-like blastopore. Molecular phylogenies however, place the acoel flatworms, which have only one opening to their digestive system, as the sister group to all remaining Bilateria. To address how this single body opening is related to the mouth and anus of the protostomes and deuterostomes, we studied the expression of genes involved in bilaterian foregut and hindgut patterning during the development of the acoel Convolutriloba longifissura. Here we show that the genes brachyury and goosecoid are expressed in association with the acoel mouth, suggesting that this single opening is homologous to the mouth of other bilaterians. In addition, we find that the genes caudal, orthopedia and brachyury-which are expressed in various bilaterian hindguts-are expressed in a small region at the posterior end of the animal, separated from the anterior oral brachyury-expressing region by a dorsal domain of ectodermal bmp2/4 expression. These results contradict the hypothesis that the bilaterian mouth and anus evolved simultaneously from a common blastoporal opening, and suggest that a through gut might have evolved independently in different animal lineages.

The great divide: septation and malformation of the cloaca, and its implications for surgeons.

The anorectal and urogenital systems arise from a common embryonic structure termed cloaca. Subsequent development leads to the division/septation of the cloaca into the urethra, urinary bladder, vagina, anal canal, and rectum. Defective cloacal development and the resulting anorectal and urogenital malformations are some of the most severe congenital anomalies encountered in children. In the most severe form in females, the rectum, vagina, and urethra fail to develop separately and drain via a single common channel known as a cloaca into the perineum. In this review, we summarize our current knowledge of embryonic cloaca development and malformation, and compare them to what has already been described in the literature. We describe the use of mouse models of cloaca malformation to understand which signaling pathways and cellular mechanisms are involved in the process of normal cloaca development. We also discuss the embryological correlation of the epithelial and stromal histology found in step sections of the common channel in 14 human cloaca malformations. Finally, we highlight the significance of these findings, compare them to prior studies, and discuss their implications for the pediatric surgeons. Understanding and identifying the molecular basis for cloaca malformation could provide foundation for tissue engineering efforts that in the future would reflect better surgical reconstruction and improved quality of life for patients.

Abnormal neural crest innervation in Sox10-Venus mice with all-trans retinoic acid-induced anorectal malformations.

BACKGROUND/PURPOSE: Despite technical advances in the surgical/medical care of anorectal malformation (ARM), persistent unsatisfactory postoperative bowel habit has been attributed to histopathologic abnormalities of the distal rectum/pouch (DRP) and hypoplasia of anal sphincter muscles (ASM). We used Sox10-Venus mice with ARM induced by all-trans retinoic acid (ATRA) to investigate neural crest cell (NCC) innervation in the DRP and ASM. METHOD: Pregnant Sox10-Venus mice were administered single doses of 50, 70, or 100 mg/kg of ATRA on embryonic day 8.5 (E8.5) then sacrificed on either E16.5 or E19.5. Bowel specimens comprising the anorectum were examined using fluorescence microscopy without immunohistochemical staining (FMIS). Anti-PGP9.5 was used to delineate ganglion cells and anti-SMA for smooth muscles. RESULTS: The appropriate dose of ATRA for inducing ARM was 50 mg/kg. Under FMIS, all ARM embryos (n = 5; all high type; 3 male:2 female) had less NCC innervation with thick Venus-positive nerve fibers in the DRP compared with normal embryos (n = 8); there was abnormal NCC innervation in the DRP and absent ASM in ARM mice. CONCLUSION: We are the first to delineate abnormal enteric nervous system innervation in the DRP of ARM mice without using immunohistochemical staining techniques thus allowing specimens to be examined without any distortion.

Rodent anogenital distance recommendations.

BACKGROUND: Measurement of rat anogenital distance (AGD) dates to at least 1912. Increased interest in endocrine disrupting chemicals and the use of AGD as a biomarker for fetal androgen effects have increased the number of studies with this endpoint in recent decades. A literature review revealed different landmarks, methods of measurement, and methods to adjust for body weight differences. AGD is often reported to hundredths of millimeters and as such, deserves precision in all these aspects. This paper presents recommendations for the measurement and analysis of rodent AGD. METHODS: Literature and regulatory guidance documents that mentioned or measured rodent AGD were reviewed. Four adjustment methods were evaluated using available online data from three rat studies each with two generations of offspring. RESULTS: Tabulation of studies reveals that species/stocks and time of data collection, but more importantly anatomical landmarks and methods of measurement have produced a variety of results which are difficult to compare. Not all studies have adjusted for test article effects on body weight (and thus size). The four adjustment methods were fairly comparable. CONCLUSION: Recommendations are as follows. A microscopic method should be used to measure AGD of late rodent fetuses and early postnatal pups. The caudal edge of the genital tubercle and the cranial edge of the anus are clear and identifiable landmarks. The simplest adjustment is to divide individual AGDs by the cube root of animals' body weight. These recommendations will help ensure data consistency and accuracy, and facilitate meaningful comparisons across laboratories and chemical classes.

A novel murine allele of Intraflagellar Transport Protein 172 causes a syndrome including VACTERL-like features with hydrocephalus.

The primary cilium is emerging as a crucial regulator of signaling pathways central to vertebrate development and human disease. We identified atrioventricular canal 1 (avc1), a mouse mutation that caused VACTERL association with hydrocephalus, or VACTERL-H. We showed that avc1 is a hypomorphic mutation of intraflagellar transport protein 172 (Ift172), required for ciliogenesis and Hedgehog (Hh) signaling. Phenotypically, avc1 caused VACTERL-H but not abnormalities in left-right (L-R) axis formation. Avc1 resulted in structural cilia defects, including truncated cilia in vivo and in vitro. We observed a dose-dependent requirement for Ift172 in ciliogenesis using an allelic series generated with Ift172(avc1) and Ift172(wim), an Ift172 null allele: cilia were present on 42% of avc1 mouse embryonic fibroblast (MEF) and 28% of avc1/wim MEFs, in contrast to >90% of wild-type MEFs. Furthermore, quantitative cilium length analysis identified two specific cilium populations in mutant MEFS: a normal population with normal IFT and a truncated population, 50% of normal length, with disrupted IFT. Cells from wild-type embryos had predominantly full-length cilia, avc1 embryos, with Hh signaling abnormalities but not L-R abnormalities, had cilia equally divided between full-length and truncated, and avc1/wim embryos, with both Hh signaling and L-R abnormalities, were primarily truncated. Truncated Ift172 mutant cilia showed defects of the distal ciliary axoneme, including disrupted IFT88 localization and Hh-dependent Gli2 localization. We propose a model in which mutation of Ift172 results in a specific class of abnormal cilia, causing disrupted Hh signaling while maintaining L-R axis determination, and resulting in the VACTERL-H phenotype.

Spatiotemporal expression of fibroblast growth factor 10 in human hindgut and anorectal development.

As fibroblast growth factor 10 (FGF-10) gene expression may have a role in anorectal duct formation, this study aimed to assess the spatiotemporal expression pattern of FGF-10 during development of the rectum and hindgut in human embryos. FGF-10 expression was evaluated in human embryos (n = 85) at 3-8 weeks of gestation after immunohistochemical evaluation using antibodies specific for FGF-10. From weeks 4 to 7 of gestation, FGF-10 expression was observed primarily in the apical epithelium of the dorsal urorectal septum, the cloacal membrane (CM) and the hindgut. Following CM rupture (week 7), the epithelium of the anal canal was negative for FGF-10; however, it was present within the urothelium through week 7. FGF-10 expression during the development of the human hindgut and anorectum suggests that it may play a role in hindgut and anorectal morphogenesis.

Anorectal malformation associated with a mutation in the P63 gene in a family with split hand-foot malformation.

PURPOSE: The aims of this study were to identify the mutation gene of a Chinese family with anorectal malformation (ARM) associated with split hand-foot malformation and to determine the spatiotemporal expression of the mutated gene during hindgut and anorectum development in human embryos. METHOD: A Chinese family with intrafamilial clinically variable manifestation was analyzed and primers were designed for exons 3-14 of P63, DLX5, DLX6, DAC, and HOXD13 as candidate genes and direct sequence analysis of the exons was performed. Immunohistochemical study of mutated gene in the hindgut and anorectum of human embryos of 4th-10th weeks was performed. RESULT: Affected individuals were found to have an Arg227Gln P63 gene mutation. From the 4th-10th weeks of gestation of the human embryo, the P63-positive cells were mainly located on the epithelium of the apical urorectal septum, hindgut, and cloacal membrane. After the anorectum ruptured during the 8th week, the P63 remained strongly immunoreactive on the epithelium of the anal canal and urethra, but the mucous membrane of the rectum exhibited no reaction. CONCLUSIONS: The mutation identified strongly suggests a causal relationship between the ARM phenotype and P63. The expression of P63 was persistently active during the dynamic and incessant septation of the cloaca and hindgut, suggesting that P63 may play a pivotal role in the morphogenesis of the hindgut and anorectum.